New microorganism and method for the production of 1.2-propanediol based on nadph dependent acetol reductase and improved nadph supply

ABSTRACT

The present invention relates to a recombinant microorganism useful for the production of 1,2-propanediol and process for the preparation of 1,2-propanediol. The microorganism of the invention is modified in a way that the 1,2-propanediol production is improved by enhancing NADPH dependent HAR activity.

FIELD OF THE INVENTION

The present invention relates to a recombinant microorganism useful forthe production of 1,2-propanediol and process for the preparation of1,2-propanediol. The microorganism of the invention is modified in a waythat the 1,2-propanediol production is improved by enhancing NADPHdependent acetol reductase activity.

BACKGROUND OF THE INVENTION

1,2-propanediol or propylene glycol, a C3 di-alcohol with formula C3H8O2or HO—CH2-CHOH—CH3, is a widely-used chemical. Its CAS number is57-55-6. It is a colorless, nearly odorless, clear, viscous liquid witha faintly sweet taste, hygroscopic and miscible with water, acetone, andchloroform. It is a component of unsaturated polyester resins, liquiddetergents, coolants, anti-freeze and de-icing fluids for aircrafts.Propylene glycol has been increasingly used since 1993-1994 as areplacement for ethylene derivatives, which are recognised as being moretoxic than propylene derivatives.

1,2-propanediol is currently produced by chemical means using apropylene oxide hydration process that consumes large amounts of water,uses highly toxic substances and generates by-products such astent-butanol and 1-phenyl ethanol.

The disadvantages of the chemical processes for the production of1,2-propanediol make biological synthesis an attractive alternative. Tworoutes have been characterized for the fermentative production of1,2-propanediol from sugars by microorganisms.

In the first route, 6-deoxy sugars (e.g. L-rhamnose or L-fucose) arecleaved into dihydroxyacetone phosphate and (S)-lactaldehyde, which canbe further reduced to (S)-1,2-propanediol (Badia et al., 1985). Thisroute is functional in E. coli, but can not yield an economicallyfeasible process due to the elevated cost of the deoxyhexoses.

The second route is the metabolism of common sugars (e.g. glucose orxylose) through the glycolysis pathway followed by the methylglyoxalpathway. Dihydroxyacetone phosphate is converted to methylglyoxal thatcan be reduced either to lactaldehyde or to hydroxyacetone (acetol).These two compounds are then transformed into 1,2-propanediol. Thisroute is used by natural producers of (R)-1,2-propanediol, such asClostridium sphenoides and Thermoanaerobacter thermosaccharolyticum.However, improvement of the performances obtained with these organismsis likely to be limited due to the lack of available genetic tools.

PRIOR ART

The methylglyoxal pathway is functional in E. coli or otherEnterobacteriaceae and several investigations for genetic modificationsof E. coli in order to obtain a 1,2-propanediol producer using simplecarbon sources have been led (WO 98/37204; Cameron et al., 1998; Altarasand Cameron, 1999; Huang et al., 1999; Altaras and Cameron, 2000;Berrios-Rivera et al., 2003; Jarboe, 2011). Improved 1,2-propanediolproducing E. coli strains obtained by a combination of rational designand evolution are described in patent applications WO 2005/073364, WO2008/116848, WO 2008/116852, WO 2008/116853, WO 2010/051849, WO2011/012693, WO 2011/012697 and WO 2011/012702, which are herebyincluded per reference.

In E. coli 1,2-propanediol producing strains, the reduction ofhydroxyacetone to 1,2-propanediol is carried out by the glyceroldehydrogenase GlyDH using NADH as a cofactor and is not total due to theinternal redox state of the cell under aerobic conditions. Aerobically,the primary role of NADH is respiratory ATP generation via oxidativephosphorylation and as a result of this, the NADH-to-NAD+ ratio isstrongly in favour of NAD+. The chemically very similar NADPH, incontrast, drives anabolic reductions, and the NADPH-to-NADP+ ratio ishigher (Fuhrer and Sauer, 2009).

Therefore it is an object of the invention to improve the production of1,2-propanediol by increasing NADPH dependent acetol reductase activityand by improving NADPH supply.

A three-dimensional structure of GlyDH from the thermophilic bacteriumBacillus stearothermophilus has been established and the NAD+ bindingsite was fully characterized (Ruzheinikov et al., 2001). A B.stearothermophilus mutant glyceraldehyde-3-phosphate able to produceNADPH has been constructed and characterized (Clermont et al., 1993).

The NADPH dependent secondary alcohol dehydrogenase (Sadh) fromClostridium beijerinckii naturally catalyzes the reduction of acetone toisopropanol (Ismaiel et al., 1993). In patent application EP2546331 fromMitsui Chemicals Inc. isopropyl alcohol is produced in an E. coli strainexpressing sadh gene from Clostridium beijerinckii.

This enzyme as well as its homologue from Thermoanaerobacter brockiihave been shown to catalyze the reduction of hydroxyacetone to1,2-propanediol and were overexpressed in the obligate photoautotrophcyanobacterium Synechococcus elongatus to produce 1,2-propanediol fromthe non-carbohydrate CO₂ as carbon source (Li and Liao, 2013).

Unexpectedly, inventors have found that overexpression of sadh genealone or in combination with other means to improve NADPH supplysignificantly improves 1,2-propanediol production from carbohydrates ascarbon source.

SUMMARY OF THE INVENTION

The invention relates to recombinant microorganisms and methods usingsaid microorganism for optimising the production of 1,2-propanediol,wherein in said microorganism the NADPH dependent acetol reductase (HAR)activity is enhanced. More particularly, in said recombinantmicroorganism, at least one gene coding for a NADPH dependent acetolreductase activity or a mutant gene gldA* coding for a NADPH dependentglycerol dehydrogenase is overexpressed, wherein said NADPH dependentacetol reductase has at least 60% amino acids identity with proteinencoded by the adh gene from Clostridium beijerinckii or the adh genefrom Thermoanaerobacter brockii or the adh1 gene from Entamoebahistolytica or the GOX1615 gene from Gluconobacter oxydans or the gld2gene from Hypocrea jecorina or the yhdN gene from Bacillus subtilis.

The recombinant microorganism used in this invention may also compriseother genetic modifications such as:

-   -   attenuated expression of gldA gene coding for a NADH dependent        glycerol dehydrogenase,    -   attenuated expression of yqhD gene coding for an aldehyde        reductase,    -   increased expression of pntAB gene operon coding for a        nicotinamide nucleotide transhydrogenase,    -   attenuated expression of pgi gene coding for a phosphoglucose        isomerase,    -   attenuated expression ofpfkA gene coding for a        phosphofructokinase,    -   increased expression of zwf gene coding for a        glucose-6-phosphate dehydrogenase,    -   increased expression of yjeF gene coding for an ADP-dependent        dehydratase,    -   increased expression of gapN gene coding for a NADP-dependent        glyceraldehyde-3-phosphate dehydrogenase,    -   increased expression of 1pd* mutant gene coding for a        NADP-dependent lipoamide dehydrogenase.

Preferably the microorganism is Escherichia coli, Klebsiella pneumoniae,Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides orSaccharomyces cerevisiae.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting, which will be limited only by the appendedclaims.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional microbiological and molecularbiological techniques within the skill of the art. Such techniques arewell known to the skilled worker, and are explained fully in theliterature.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “amicroorganism” includes a plurality of such microorganisms, and areference to “an endogenous gene” is a reference to one or moreendogenous genes, and so forth. Unless defined otherwise, all technicaland scientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any materials and methods similar or equivalent tothose described herein can be used to practice or test the presentinvention, the preferred materials and methods are now described.

In the claims that follow and in the consecutive description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise”, “contain”,“involve” or “include” or variations such as “comprises”, “comprising”,“containing”, “involved”, “includes”, “including” are used in aninclusive sense, i.e. to specify the presence of the stated features butnot to preclude the presence or addition of further features in variousembodiments of the invention.

The present invention is related to a method for the production of1,2-propanediol in a fermentative process comprising the steps ofculturing a microorganism genetically modified for the production of1,2-propanediol in an appropriate culture medium comprising acarbohydrate as source of carbon and recovering 1,2-propanediol from theculture medium, wherein the NADPH dependent HAR activity is enhanced insaid genetically modified microorganism. Particularly preferredembodiments of the invention are further described below.

The terms “acetol reductase” and “hydroxyacetone reductase” or “HAR” areused interchangeably and denote enzymatic activity of reduction ofacetol (or hydroxyacetone) into 1,2-propanediol. This activity may beNADPH dependent or NADH dependent.

The term “microorganism”, as used herein, refers to a bacterium, yeastor fungus which is not modified artificially. Preferentially, themicroorganism is selected among Enterobacteriaceae, Bacillaceae,Clostridiaceae, Streptomycetaceae and yeast. More preferentially themicroorganism is a species of Escherichia, Klebsiella,Thermoanaerobacterium, Clostridium or Saccharomyces. Even morepreferentially the microorganism is selected among Escherichia coli,Klebsiella pneumoniae, Thermoanaerobacterium thermosaccharolyticum,Clostridium sphenoides or Saccharomyces cerevisiae. Preferentially, themicroorganism is a heterotroph microorganism, i.e. is not capable offixing atmospheric carbon and uses instead organic carbon sources forits growth. Even more preferably, the heterotroph microorganism of theinvention is Escherichia coli.

The term “recombinant microorganism” or “genetically modifiedmicroorganism”, as used herein, refers to a bacterium, yeast or fungusthat is not found in nature and is genetically different from itsequivalent found in nature. It means, it is modified either byintroduction or by deletion or by modification of genetic elements. Itcan also be transformed by forcing the development and evolution of newmetabolic pathways by combining directed mutagenesis and evolution underspecific selection pressure (see, for example, WO2005/073364 orWO2008/116852).

A microorganism may be modified to express exogenous genes if thesegenes are introduced into the microorganism with all the elementsallowing their expression in the host microorganism. The modification or“transformation” of microorganisms with exogenous DNA is a routine taskfor those skilled in the art.

A microorganism may be modified to modulate the expression level of anendogenous gene.

The term “endogenous gene” means that the gene was present in themicroorganism before any genetic modification. Endogenous genes may beoverexpressed by introducing heterologous sequences in addition to, orto replace endogenous regulatory elements, or by introducing one or moresupplementary copies of the gene into the chromosome or a plasmid.Endogenous genes may also be modified to modulate their expressionand/or activity. For example, mutations may be introduced into thecoding sequence to modify the gene product or heterologous sequences maybe introduced in addition to or to replace endogenous regulatoryelements. Modulation of an endogenous gene may result in theup-regulation and/or enhancement of the activity of the gene product, oralternatively, down regulate and/or lower the activity of the endogenousgene product.

Another way to modulate their expression is to exchange the endogenouspromoter of a gene (e.g., wild type promoter) with a stronger or weakerpromoter to up or down regulate expression of the endogenous gene. Thesepromoters may be homologous or heterologous. It is well within theability of the person skilled in the art to select appropriatepromoters.

In contrast, “exogenous gene” means that the gene was introduced into amicroorganism, by means well known by the man skilled in the art whereasthis gene is not naturally occurring in the microorganism. Exogenousgenes may be integrated into the host chromosome, or be expressedextra-chromosomally by plasmids or vectors. A variety of plasmids, whichdiffer with respect to their origin of replication and their copy numberin the cell, are well known in the art. These genes may be homologous.“Overexpression” or “overexpressing” is also used to designateexpression of exogenous genes in the microorganisms.

In the context of the invention, the term “homologous gene” is notlimited to designate genes having a theoretical common genetic ancestor,but includes genes which may be genetically unrelated that have, nonethe less, evolved to encode protein which perform similar functionsand/or have similar structure. Therefore the term ‘functional homolog”for the purpose of the present invention relates to the fact that acertain enzymatic activity may not only be provided by a specificprotein of defined amino acid sequence, but also by proteins of similarsequence from other (un)related microorganisms.

Using the references given in Uniprot for known protein or in Genbankfor known genes, those skilled in the art are able to obtain proteinand/or gene sequences and to determine the equivalent genes in otherorganisms, bacterial strains, yeast, fungi, mammals, plants, etc. Thisroutine work is advantageously done using consensus sequences that canbe determined by carrying out sequence alignments with genes derivedfrom other microorganisms and designing degenerate probes to clone thecorresponding gene in another organism. These routine methods ofmolecular biology are well known to those skilled in the art.

According to the invention, the terms “fermentative process’,‘fermentation” or ‘culture’ are used interchangeably to denote thegrowth of microorganism. This growth is generally conducted infermenters with an appropriate growth medium adapted to themicroorganism being used.

An “appropriate culture medium” designates a medium (e.g., a sterile,liquid media) comprising nutrients essential or beneficial to themaintenance and/or growth of the cell such as carbon sources or carbonsubstrates, nitrogen sources, for example, peptone, yeast extracts, meatextracts, malt extracts, urea, ammonium sulfate, ammonium chloride,ammonium nitrate and ammonium phosphate; phosphorus sources, forexample, monopotassium phosphate or dipotassium phosphate; traceelements (e.g., metal salts), for example magnesium salts, cobalt saltsand/or manganese salts; as well as growth factors such as amino acidsand vitamins.

The term “source of carbon”, “carbon source” or “carbon substrate”according to the present invention refers to any carbon source capableof being metabolized by a microorganism wherein the substrate containsat least one carbon atom.

The term “carbohydrate” refers to any carbon source capable of beingmetabolized by a microorganism and containing at least one carbon atom,two atoms of hydrogen and one atom of oxygen. CO₂ is not a carbohydratebecause it does not contain hydrogen.

The carbohydrate is selected among the group consisting ofmonosaccharides such as glucose, fructose, mannose, xylose, arabinose,galactose and the like, disaccharides such as sucrose, cellobiose,maltose, lactose and the like, oligosaccharides such as raffinose,stacchyose, maltodextrins and the like, polysaccharides such ascellulose, hemicellulose, starch and the like, methanol, formaldehydeand glycerol. Especially preferred carbon sources are arabinose,fructose, galactose, glucose, lactose, maltose, sucrose, xylose or amixture thereof. More preferably carbon source is sucrose.

In a particular embodiment of the invention, the carbon source isderived from renewable feed-stock. Renewable feed-stock is defined asraw material required for certain industrial processes that can beregenerated within a brief delay and in sufficient amount to permit itstransformation into the desired product. Vegetal biomass treated or not,is an interesting renewable carbon source.

Those skilled in the art are able to define the culture conditions forthe microorganisms according to the invention. In particular thebacteria are fermented at a temperature between 20° C. and 55° C.,preferentially between 25° C. and 40° C., and more specifically about30° C. to 37° C. for E. coli.

This process can be carried out either in a batch process, in afed-batch process or in a continuous process. It can be carried outunder aerobic, micro-aerobic or anaerobic conditions.

‘Under aerobic conditions’ means that oxygen is provided to the cultureby dissolving the gas into the liquid phase. This could be obtained by(1) sparging oxygen containing gas (e.g. air) into the liquid phase or(2) shaking the vessel containing the culture medium in order totransfer the oxygen contained in the head space into the liquid phase.The main advantage of the fermentation under aerobic conditions is thatthe presence of oxygen as an electron acceptor improves the capacity ofthe strain to produce more energy under the form of ATP for cellularprocesses. Therefore the strain has its general metabolism improved.

Micro-aerobic conditions are defined as culture conditions wherein lowpercentages of oxygen (e.g. using a mixture of gas containing between0.1 and 10% of oxygen, completed to 100% with nitrogen), is dissolvedinto the liquid phase.

Anaerobic conditions are defined as culture conditions wherein no oxygenis provided to the culture medium. Strictly anaerobic conditions areobtained by sparging an inert gas like nitrogen into the culture mediumto remove traces of other gas. Nitrate can be used as an electronacceptor to improve ATP production by the strain and improve itsmetabolism.

The phrase “recovering 1,2-propanediol from the culture medium”designates the process of purifying the produced 1,2-propanediol, usingmethods known by the man skilled in the art. Such methods are disclosedespecially in patent applications WO2011/076690 and WO2012/130316.

The terms “microorganism genetically modified for the production of1,2-propanediol” refer to microorganisms modified through either theintroduction or deletion of genetic elements, or through a step ofevolution as described in patent application WO 2005/073364. Inparticular, it designates a genetically modified microorganismpresenting an improved 1,2-propanediol production in comparison with theendogenous production of the corresponding wild-type microorganism,without genetic modifications. Such microorganism is for exampledescribed in the patent application WO 2008/116848, WO 2008/116853, WO2011/012693, WO 2011/012697, WO 2011/012702 or EP2532751 incorporated byreference. Preferred genetic modifications are the following:

-   -   increased expression of at least one gene selected among mgsA        gene encoding methylglyoxal synthase, yqhD, yafB, ycdW, yqhE,        yeaE, yghZ, yajO, tas, ydjG or ydbC genes encoding methylglyoxal        reductase, gldA gene encoding glycerol dehydrogenase and fuc0        gene encoding lactaldehyde reductase;    -   deletion of either the edd gene encoding phosphphogluconate        dehydratase or eda gene encoding 2-keto-3-deoxygluconate        6-phosphate aldolase or both;    -   attenuation of the synthesis of unwanted by-products by deletion        of the genes coding for enzymes involved in synthesis of lactate        from methylglyoxal (such as gloA gene encoding glyoxalase I,        aldA gene encoding aldehyde dehydrogenase A, aldB gene encoding        acetaldehyde dehydrogenase), lactate from pyruvate (ldhA gene        encoding lactate dehydrogenase), formate (pflA gene encoding        pyrvate formate lyase, pflB gene encoding pyruvate formate        lyase), ethanol (adhE gene encoding aldehyde-alcohol        dehydrogenase) and acetate (ackA gene encoding acetate kinase,        pta gene encoding phosphate acetyltransferase, poxB gene        encoding pyruvate oxidase);    -   elimination of the pathways consuming PEP like pyruvates kinases        (encoded by the pykA and pykF genes) and/or by promoting the        synthesis of PEP e. g. by overexpressing the ppsA gene coding        for PEP synthase;    -   specific mutation in the lpd gene encoding lipoamide        dehydrogenase;    -   the arcA gene encoding ArcA transcriptional dual regulator and        the ndh gene encoding NADH:ubiquinone oxidoreductase II can be        deleted,    -   the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase        is under the control of temperature inducible promoter,    -   genes involved in the importation and metabolism of sucrose        (cscB gene encoding sucrose permease, cscA gene encoding sucrose        hydrolase, cscK gene encoding fructokinase, scrA gene encoding        Enzyme II of the phosphoenolpyruvate-dependent        phosphotransferase system, scrK gene encoding ATP-dependent        fructokinase, scrB gene encoding sucrose 6-phosphate hydrolase        (invertase), scrY gene encoding sucrose porine) are added or        their expression is increased.

A preferred genetic modification is the improvement of methylglyoxalreductase activity, obtained by an increased expression of the geneyqhD*(G149E).

Another preferred genetic modification is the improvement ofmethylglyoxal synthase activity, obtained by an increased expression ofthe gene mgsA*(H21Q).

Another preferred genetic modification is the improvement ofhydroxyacetone reductase activity, obtained by an increased expressionof the gene gldA*(A160T). This HAR mutant is NADH dependent unlike theHAR mutant NADPH dependent disclosed hereinafter.

Hydroxyacetone reductase (HAR) is the last enzyme involved in theproduction of 1,2-propanediol. HAR catalyzes the following reaction:

Hydroxyacetone+NAD(P)H→1,2-propanediol+NAD(P)⁺

A 1,2-propanediol producing strain wherein the gldA gene is deleted isnot able to produce 1,2-propanediol anymore and accumulateshydroxyacetone (WO2008/116851). The GldA protein has been purified tohomogeneity and uses NADH as a cofactor (Kelley and Dekker, 1984). Inaerobic conditions, the conversion of hydroxyacetone to 1,2-propanediolis not total due to the internal redox state of the cell : under suchconditions, the primary role of NADH is respiratory ATP generation viaoxidative phosphorylation and as a result of this, the NADH-to-NAD+ratio is strongly in favour of NAD+. The chemically very similar NADPH,in contrast, drives anabolic reductions, and the NADPH-to-NADP+ ratio ishigher (Fuhrer and Sauer, 2009).

Following this observation, the object of this invention is to improvethe production of 1,2-propanediol by increasing NADPH dependenthydroxyacetone reductase activity.

Increasing an activity can be obtained by improving the proteincatalytic efficiency or decreasing protein turnover or decreasingmessenger RNA (mRNA) turnover or increasing transcription of the gene orincreasing translation of the mRNA.

Improving the protein catalytic efficiency means increasing the kcatand/or decreasing the Km for a given substrate and/or a given cofactor,and/or increasing the Ki for a given inhibitor. kcat, Km and Ki areMichaelis-Menten constants that the man skilled in the art is able todetermine (Segel, 1993). Decreasing protein turnover means stabilizingthe protein. Methods to improve protein catalytic efficiency and/ordecrease protein turnover are well known from the man skilled in theart. Those include rational engineering with sequence and/or structuralanalysis and directed mutagenesis, as well as random mutagenesis andscreening. Mutations can be introduced by site-directed mutagenesis byusual methods like Polymerase Chain Reaction (PCR), or by randommutagenesis techniques, such as use of mutagenic agents (Ultra-Violetrays or chemical agents like nitrosoguanidine (NTG) orethylmethanesulfonate (EMS)) or use of PCR techniques (DNA shuffling orerror-prone PCR). Stabilizing the protein can also be achieved by addinga peptide sequence called “tag” either at the N-terminus or theC-terminus of the protein. Tags are well known from the man skilled inthe art. For instance, a Glutathione-S-Transferase (GST) can be used tostabilize a protein.

Decreasing mRNA turnover can be achieved by modifying the gene sequenceof the 5′-untranslated region (5 ‘-UTR) and/or the coding region, and/orthe 3’-UTR (Carrier and Keasling, 1999).

Increasing transcription of a gene can be achieved by increasing thenumber of copies of the gene and/or using a promoter leading to a higherlevel of expression of the gene. “Overexpression” or “overexpressing” isalso used to designate increasing transcription of a gene in themicroorganisms.

For increasing the number of copies of the gene in the microorganism,the gene is encoded chromosomally or extrachromosomally. When the geneis located on the chromosome, several copies of the gene can beintroduced on the chromosome by methods of recombination, known by theexpert in the field (including gene replacement). When the gene islocated extra-chromosomally, it may be carried by different types ofplasmids that differ with respect to their origin of replication andthus their copy number in the cell. These plasmids are present in themicroorganism in 1 to 5 copies, or about 20 copies, or up to 500 copies,depending on the nature of the plasmid: low copy number plasmids withtight replication (e.g for E.coli pSC101, RK2), low copy number plasmids(e.g for E. coli pACYC, pRSF1010) or high copy number plasmids (e.g forE.coli pSK bluescript II).

For using a promoter leading to a high level of expression of the genethe man skilled in the art knows which promoters are the mostconvenient, for example promoters Ptrc, Ptac, Plac, or the lambdapromoter cI are widely used. These promoters can be “inducible” by aparticular compound or by specific external condition like temperatureor light. These promoters may be homologous or heterologous.

Increasing translation of the mRNA can be achieved by modifying theRibosome Binding Site (RBS). A RBS is a sequence on mRNA that is boundby the ribosome when initiating protein translation. It can be eitherthe 5′ cap of a mRNA in eukaryotes, a region 6-7 nucleotides upstream ofthe start codon AUG in prokaryotes (called the Shine-Dalgarno sequence),or an internal ribosome entry site (IRES) in viruses. By modifying thissequence, it is possible to change the protein translation initiationrate, proportionally alter its production rate, and control its activityinside the cell. The same RBS sequence will not have the same impactaccording to the nature of the mRNA. It is possible to optimize thestrength of a RBS sequence to achieve a targeted translation initiationrate by using the software RBS CALCULATOR (Salis, 2011).

In a first aspect of the present invention, the production of1,2-propanediol is improved by enhancing NADPH dependent acetolreductase (NADPH HAR) activity in the modified microorganism.

In a first embodiment of the invention, NADPH HAR activity is enhancedby overexpressing a gene encoding a NADPH HAR (NADPH HAR gene) in themodified microorganism.

Adh from Clostridium beijerinckii naturally catalyzes the reduction ofacetone to isopropanol (Ismaiel et al., 1993). This enzyme as well asits homologue from Thermoanaerobacter brockii have been shown tocatalyze the reduction of hydroxyacetone to 1,2-propanediol and wereoverexpressed in the obligate photoautotroph cyanobacteriumSynechococcus elongatus to produce 1,2-propanediol from CO₂ as carbonsource (Li and Liao, 2013).

Data mining and sequence analysis have been used to determine somecandidate homologous enzymes and genes coding for such enzymes. Thesecandidates are disclosed in Table 1 below.

The means of identifying homologous sequences and their percentagehomologies are well known to those skilled in the art, and include inparticular the BLAST programs. The sequences obtained can then beexploited (e.g., aligned) using, for example, the programs CLUSTALW orMULTALIN. Another way to identify protein homologues is through theconstruction of phylogenetic trees, using bioinformatics programs wellknown by the man skilled in the art.

TABLE 1 Possible candidates for NADPH dependent HAR enzymes: OrganismFunction UNIPROT Mycoplasma pneumoniae NADP-dependent isopropanoldehydrogenase P75214 Entamoeba histolytica NADP-dependent isopropanoldehydrogenase P35630 Gadus callarias Probable NADP-dependent isopropanoldehydrogenase P26325 Photobacterium damselae NADP-dependent isopropanoldehydrogenase P39450 Synechocystis sp Alcohol dehydrogenase 1 P73138Oryza sativa S-(hydroxymethyl)glutathione dehydrogenase Q4R1E8 Hordeumvulgare S-(hydroxymethyl)glutathione dehydrogenase P10848 Sparus aurataAlcohol dehydrogenase 2 P79896 Aspergillus nidulans NADP(+)-dependentglycerol dehydrogenase Q7Z8L1 Gadus morhua Alcohol dehydrogenase 3P81600 Hordeum vulgare Alcohol dehydrogenase class-3 P10847 Zea maysAlcohol dehydrogenase class-3 chain H P04707 Trichomonas vaginalisNADP-dependent alcohol dehydrogenase, putative A2EU60 Saccharomycescerevisiae Alcohol dehydrogenase 2 P38230 Pennisetum glaucum Alcoholdehydrogenase 2 P14219 Oryctolagus cuniculus Probable quinoneoxidoreductase O46650 Arabidopsis thaliana Alcohol dehydrogenase 1Q0V7W6 Uromastyx hardwickii Alcohol dehydrogenase class-2 isozyme 2P25405 Amycolatopsis methanolica Alcohol dehydrogenase-like 5 P80094 Zealuxurians Alcohol dehydrogenase 1A Q07264 Gluconobacter oxydans Aldehydereductase Q5FQJ0 Zea mays S-(hydroxymethyl)mycothiol dehydrogenaseP00333 Oryctolagus cuniculus Alcohol dehydrogenase 1 O46649 Arabidopsisthaliana Alcohol dehydrogenase 1 P06525 Arabidopsis thaliana Alcoholdehydrogenase class-2 isozyme 1 Q8LEB2 Cupriavidus necator Alcoholdehydrogenase class-P P14940 Rana perezi Alcohol dehydrogenase-like 6P22797 Arabidopsis thaliana Alcohol dehydrogenase Q9SK87 Rattusnorvegicus Alcohol dehydrogenase 1 Q64563 Malus domestica Alcoholdehydrogenase-like 2 P48977 Saccharomyces _(—) cerevisiae Glycerol2-dehydrogenase (NADP(+)) P14065 Drosophila melanogaster Alcoholdehydrogenase 4 P46415 Mus musculus Alcohol dehydrogenase Q9QYY9 Homosapiens Alcohol dehydrogenase class-3 P08319 Bacillus subtilis Alcoholdehydrogenase 4 O31776 Peromyscus maniculatus Alcohol dehydrogenase 4P41681 Hansenula polymorpha Glycerol dehydrogenase Q6BC32 Bacilluslicheniformis L-threonine 3-dehydrogenase Q65JE7 Arabidopsis thalianaAlcohol dehydrogenase 6 Q9SK86 Aspergillus fumigatus L-threonine3-dehydrogenase B0YC65 Saccharomyces cerevisiae Alcoholdehydrogenase-like 1 P32771 Staphylococcus aureus Probable D-xylulosereductase A Q6GBM4 Staphylococcus aureus S-(hydroxymethyl)glutathionedehydrogenase Q7A742 Hypocrea jecorina General stress protein 69 Q0GYU4Aspergillus oryzae Alcohol dehydrogenase Q86ZV0 Aspergillus clavatusAlcohol dehydrogenase A1CFY8 Sulfolobus tokodaii D-xylulose reductase AQ96XE0 Neosartorya fischeri Probable D-xylulose reductase A A1D9C9Metallosphaera sedula NAD-dependent alcohol dehydrogenase A4YGN0Sulfolobus sp Probable D-xylulose reductase A P50381 Bacillus subtilisSuccinate-semialdehyde dehydrogenase (acetylating) Q06004 Escherichiacoli NAD-dependent alcohol dehydrogenase P77280 Saccharomyces cerevisiaeSorbitol dehydrogenase Q07993 Thermococcus kodakarensis Uncharacterizedzinc-type alcohol dehydrogenase-like Q5JI69 protein Methylobactermarinus D-xylulose reductase P47734 Bacillus subtilis ProbableL-threonine 3-dehydrogenase O35045 Bacillus subtilisS-(hydroxymethyl)glutathione dehydrogenase O06012 Schizosaccharomycespombe Uncharacterized zinc-type alcohol dehydrogenase-like Q9P6I8protein Dunaliella salina Dihydroxyacetone reductase B8Y210 Escherichiacoli Uncharacterized zinc-type alcohol dehydrogenase-like P75691 proteinMycobacterium tuberculosis Zinc-type alcohol dehydrogenase-like proteinO07737 Saccharomyces cerevisiae Aldehyde reductase Q04894 Streptomycestenebrarius Probable zinc-binding alcohol dehydrogenase Q2MF22Escherichia coli NADP-dependent alcohol dehydrogenase 6 P27250 Bacillussubtilis 2-deoxy-scyllo-inosamine dehydrogenase P80874

In this embodiment of the invention, the gene encoding a NADPH HARoverexpressed in the recombinant microorganism encodes a NADPH dependentacetol reductase chosen among the proteins listed above. From Uniprotnumber, the man skilled in the art is able to obtain number and sequenceof corresponding gene. Preferentially, the microorganism of theinvention expresses at least one gene coding for a NADPH dependentacetol reductase (NADPH HAR gene) having at least 60%, preferably atleast 70%, more preferably at least 85% and even more preferably atleast 90% amino acids identity with complete protein encoded by genesadh from Clostridium beijerinckii or adh from Thermoanaerobacter brockiior adh1 from Entamoeba histolytica or GOX1615 from Gluconobacter oxydansor gld2 from Hypocrea jecorina or yhdN from Bacillus subtilis. Morepreferentially, the microorganism of the invention expresses a genechosen among adh from Clostridium beijerinckii or adh fromThermoanaerobacter brockii or adh1 from Entamoeba histolytica or GOX1615from Gluconobacter oxydans r gld2 from Hypocrea jecorina or yhdN fromBacillus subtilis (i.e. 100% amino acids sequence identity with theprotein sequence encoded by said genes). Even more preferentially, theNADPH dependent acetol reductase is encoded by adh gene from Clostridiumbeijerinckii.

Sequence identity between amino acid sequences can be determined bycomparing a position in each of the sequences which may be aligned forthe purposes of comparison. When a position in the compared sequences isoccupied by the same amino acid, then the sequences are identical atthat position. A degree of sequence identity between proteins is afunction of the number of identical amino acid residues at positionsshared by the sequences of said proteins.

To determine the percentage of identity between two amino acidsequences, the sequences are aligned for optimal comparison. Forexample, gaps can be introduced in the sequence of a first amino acidsequence for optimal alignment with the second amino acid sequence. Theamino acid residues at corresponding amino acid positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue as the corresponding position in the second sequence,the molecules are identical at that position.

The percentage of identity between the two sequences is a function ofthe number of identical positions shared by the sequences. Hence %identity=number of identical positions/total number of overlappingpositions X 100.

Optimal alignment of sequences may be conducted by the global homologyalignment algorithm of Needleman and Wunsch (1970), by computerizedimplementations of this algorithm or by visual inspection. The bestalignment (i.e., resulting in the highest percentage of identity betweenthe compared sequences) generated by the various methods is selected.

In other words, the percentage of sequence identity is calculated bycomparing two optimally aligned sequences, determining the number ofpositions at which the identical amino acid occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions and multiplying the result by100 to yield the percentage of sequence identity.

In another preferred embodiment of the invention, NADPH HAR activity isenhanced by increasing NADPH dependent HAR activity as described aboveand/or by decreasing NADH dependent HAR activity.

Decreasing the activity of an enzyme means either decreasing itsspecific catalytic activity by mutating the gene so as to change theamino acid sequence and/or decreasing concentrations of the protein inthe cell by mutating the nucleotidic sequence or by deleting the codingregion of the gene.

In the present invention, decrease in NADH dependent HAR activity wasperformed by deleting the gldA gene, or by performing cofactorengineering on GldA enzyme, which resulted both in a decrease of NADHdependent HAR activity and in an increase of NADPH dependent HARactivity.

Therefore, the microorganism of the invention preferably overexpresses amutant gldA*gene coding for a NADPH dependent glycerol dehydrogenaseand/or is deleted in endogenous gldA gene.

Cofactor engineering means changing the cofactor specificity of anenzyme by replacing some specific amino acid residues in GldA protein.These resulted mutants (G1dA*NADPH) are NADPH dependent and aredifferent from GldA*mutant (G1dA*A160T) disclosed above which is NADHdependent. The residues governing cofactor specificity in thecofactor-binding pocket known as the ‘Rossman fold’ have been thoroughlystudied (Scrutton et al., 1990; Clermont et al., 1993; Corbier et al.,1990; Wu et al., 2012) and the man skilled in the art is able to definewhich amino acid residues to modify in order to change cofactorspecificity. Cofactor engineering has recently been successful inaltering enzymes to prefer NADH as a cofactor instead of NADPH. Anexample is the enzyme Gre2p, an NADPH-preferring dehydrogenase found inSaccharomyces cerevisiae, which was modified by direct mutagenesis tohave a decreased dependency on NADPH, and an increased affinity for NADH(Katzberg et al., 2010).

In the present invention a tri-dimensional structure model of GldA fromE. coli was built from the tri-dimensional structure of the enzyme fromBacillus stearothermophilus (Ruzheinikov et al., 2001). The man skilledin the art knows how to build tri-dimensional homology models using forexample a software such as Discovery Studio (Accelrys). Such models areuseful for determining which amino acid residues to mutate in order toachieve the desired change in cofactor specificity. From thetri-dimensional structure of GldA from E. coli the inventors haveidentified amino acid residues to be replaced by a different amino acidresidue at the same position:

-   -   D37    -   F39    -   V40    -   F43    -   T116    -   P161    -   L164

According to the invention, the change in GldA cofactor specificity ismediated by at least one mutation at position D37. In a preferredembodiment, the amino acid residue at position D37 is replaced by aglycine (D37G), an alanine (D37A) or a valine (D37V). In a mostpreferred embodiment, the amino acid residue at position D37 is replacedby a glycine (D37G).

In a preferred embodiment, the change in GldA cofactor specificity isimproved by combining a mutation at position D37 with at least onemutation at position P161. Preferentially, the amino acid residue atposition P161 is replaced by a serine (P161S) or a threonine (P161T).More preferentially, the amino acid residue at position P161 is replacedby a serine (P161S).

In a most preferred embodiment, the change in GldA cofactor specificityis improved by combining mutations at positions D37 and P161 with atleast one mutation at position L164. Preferentially, the amino acidresidue at position L164 is replaced by an alanine (L164A), a glycine(L164G) or a valine (L164V). More preferentially the amino acid residueat position L164 is replaced by an alanine (L164A).

In a specific embodiment, the microorganism of the inventionoverexpresses a mutant gldA*encoding GldA*mutant containing at least thefollowing mutations: D37G, P161S and L164A.

In a preferred embodiment of the invention these mutations may beintroduced in the GldA*mutant previously constructed and containingmutation A160T.

The amino acid sequence of the glycerol dehydrogenase expressed byE.coli (strain K12) is publicly available on the UniprotKB databaseunder the reference P0A9S5.

Hence, in order to enhance NADPH HAR dependent actitivy, themicroorganism of the invention preferably overexpresses at least onegene coding for a NADPH dependent acetol reductase (NADPH HAR) asdescribed above or a mutant gldA*gene coding for a NADPH dependentglycerol dehydrogenase as described above.

In other specific embodiments of the invention, the modifiedmicroorganism of the invention may:

-   -   overexpress at least one NADPH HAR gene and may be deleted in        gldA gene,    -   overexpress at least one NADPH HAR gene and gldA*NADPH mutant,        or    -   overexpress at least one NADPH HAR gene and gldA*NADPH mutant,        and may be deleted in gldA gene.

In another aspect of the invention, the production of 1,2-propanediol isfurther improved by combining an increase in NADPH dependent HARactivity as described above with an increase in NADPH availability inthe cell.

Strategies for increasing NADPH availability in the cell are well knownfrom the man skilled in the art (reviewed in Lee et al., 2013). In thepresent invention, NADPH availability in the cell is increased by:

-   -   overexpressing the pntAB operon coding for the membrane-bound        transhydrogenase (WO2012/055798A1), and/or    -   attenuating the pgi gene coding for a phosphoglucose isomerase,        and/or    -   increasing the flux through the pentose phosphate pathway by        decreasing the activity of the phosphofructokinase encoded by        pfkA gene (WO2005/047498) and overexpressing the zwf gene coding        for the glucose-6-phosphate dehydrogenase (Shin et al., 2002),        and/or    -   overexpressing the gapN gene from Streptococcus mutans coding        for the

NADPH generating glyceraldehyde-3-phosphate (Centeno-Leija et al.,2013),

-   -   and/or overexpressing a mutant lpd*gene coding for a lipoamide        dehydrogenase able to generate NADPH (Bocanegra et al., 1993),    -   and/or overexpressing the yjeF gene coding for an ADP-dependent        dehydratase reactivating NADH(X) and NADPH(X) produced by        enzymatic or heat-dependent hydration (Marbaix et al., 2011).

In a further aspect of the invention, when the microorganism of theinvention overexpresses NADPH HAR gene, the yqhD gene coding formethylglyoxal reductase (MGR) has been deleted. Under such conditions,the HAR protein not only acts as a NADPH dependent HAR enzyme but alsoas a NADPH dependent MGR enzyme, carrying the two-step NADPH dependentreduction of methylglyoxal into 1,2-propanediol. Such activity may becarried out by any of the candidates for NADPH dependent HAR enzymesthat have been listed above.

In a more preferred embodiment of the invention, the production of1,2-propanediol in a fermentative process by a recombinantmicroorganism, wherein NADPH dependent acetol reductase activity isenhanced according to any of the modifications described above, fromcarbohydrate as carbon source, may be achieved through a combination ofthe above discussed modifications in said microorganism, for example:

-   -   expressions of at least one NADPH HAR gene and pntAB genes are        enhanced,    -   expression of at least one NADPH HAR gene is enhanced and gldA        gene is deleted,    -   expressions of at least one NADPH HAR gene and pntAB are        enhanced whereas gldA gene is deleted,    -   expressions of at least one NADPH HAR gene and yjeF are enhanced        whereas gldA gene is deleted,    -   expressions of at least one NADPH HAR gene is enhanced and pgi        is attenuated whereas gldA gene is deleted,    -   expressions of at least one NADPH HAR gene and zwf are enhanced        whereas gldA gene and pfkA gene are attenuated,    -   expressions of at least one NADPH HAR gene and gapN are enhanced        whereas gldA gene is deleted,    -   expressions of at least one NADPH HAR gene and lpd*mutant are        enhanced whereas gldA gene is deleted    -   expressions of a mutant GldA*NADPH encoding gene are enhanced        and gldA gene is deleted,    -   expressions of a mutant GldA*NADPH encoding gene and pntAB genes        are enhanced,    -   expressions of a mutant GldA*NADPH encoding gene and pntAB genes        are enhanced whereas gldA gene is deleted.    -   expressions of at least one NADPH HAR gene is enhanced and yqhD        gene is attenuated whereas gldA gene is deleted    -   expressions of at least one NADPH HAR gene and pntAB genes are        enhanced whereas yqhD gene and gldA gene are deleted.

EXAMPLES Example 1 Methods

In the examples given below, methods well known in the art were used toconstruct E. coli strains containing replicating vectors and/or variouschromosomal deletions, and substitutions using homologous recombinationwell described by Datsenko & Wanner, (2000) for Escherichia coli. In thesame manner, the use of plasmids or vectors to express or overexpressone or several genes in a recombinant microorganisms are well known bythe man skilled in the art. Examples of suitable E. coli expressionvectors include pTrc, pACYC184n pBR322, pUC18, pUC19, pKC30, pRep4,pHS1, pHS2, pPLc236, etc. . . .

Several protocols have been used in the following examples. Protocol 1(chromosomal modifications by homologous recombination, selection ofrecombinants), protocol 2 (transduction of phage P1) and protocol 3(antibiotic cassette excision, the resistance genes were removed whennecessary) used in this invention have been fully described in patentapplication EP 2532751. Chromosomal modifications were verified by a PCRanalysis with appropriate oligonucleotides that the person skilled inthe art is able to design.

Protocol 4: Construction of Recombinant Plasmids

Recombinant DNA technology is well described in the art. The DNAfragments and chosen plasmid were digested with compatible restrictionenzymes (that the person skilled in the art will be able to define),then ligated and transformed into competent cells. Transformants wereanalysed and recombinant plasmids of interest were verified by DNAsequencing.

TABLE 2 Sequences cited in the following examples SEQ ID NoSequence 5′→3′  1 cgtgcaaacctacaagccgatcttgccattgtaggcgccggtggcgcgggattacgtgctgcaattgctgccgcgcaggcCATATGAATA TCCTCCTTAG  2CGTTAGATTGTAACGACACCAATCAGCGTGACAACTGTCAGGATAGCAGCCAGACCGTAGAAAACCCATTTGCCCGCAGGTGTAGGCTGG AGCTGCTTCG  3ATGTTTAAGAATGCATTTGCTAACCTGCAAAAGGTCGGTAAATCGCTGATGCTGCCGGTATCCGTACTGCCTATCGCAGGtgtaggctgg agctgcttcg  4TTAGTGGTTACGGATGTACTCATCCATCTCGGTTTTCAGGTTATCGGATTTAGTACCGAAAATCGCCTGAACACCAGAACcatatgaata tcctccttag  5atggaccgcattattcaatcaccgggtaaatacatccagggcgctgatgtgattaatcgtctgggcgaatacctgaagccgtgtaggctg gagctgcttcg  6TTATTCCCACTCTTGCAGGAAACGCTGACCGTACTGGTCGGCTACCAGCAGAGCGGCGTAAACCTGATCTGGCGTCGCGCCATATGAATA TCCTCCTTAG  7ATCCGGTATAGGAGGTATAGA  8 gaccgtcgaagacaattatcagtctttatccggcgttctaaggtgtttatcccactatcacggctgaatcgttaatattttgcgagttcacgccgaaatactgatttaggcgctagatcacaggcataattttcagtacgttatagggcgtttgttactaatttattttaacggagtaacatttagctcgtacatgagcagcttgtgtggctcctgacacaggcaaaccatcatcaataaaaccgatggaagggaatatc  9atgcgaattggcataccaagagaacggttaaccaatgaaacccgtgttgcagcaacgccaaaaacagtggaacagctgctgaaactgggttttaccgtcgcggtagagagcggcgcgggtcaactggcaagattgacgataaagcgtagtgcaagcgggcgctgaaattgtagaagggaatagcgtctggcagtcagagatcattctgaaggtcaatgcgccgttagatgatgaaattgcgttactgaatcctgggac 10 AGACAATAATCGAACAACATATTAAGGAGAGTTT11 ccaacgcagaccgctgcctggcaggcactacagaaacacttcgatgaaatgaaagacgttacgatcgccgatctttttgctgtaggctgg agctgcttcg 12GCGCCACGCTTTATAGCGGTTAATCAGACCATTGGTCGAGCTATCGTGGCTGCTGATTTCTTTATCATCTTTCAGCTCTGCATATGAATA TCCTCCTTAG 13gttcctcggttctgcgcgtttcccggaattccgcgacgagaacatccgcgccgtggctatcgaaaacctgaaaaaacgtggtgtaggctg gagctgcttcg 14GGCCTGATAAGCGAAGCGCATCAGGCATTTTTGCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCACATATGAA TATCCTCCTTAG 15gttcctcggttctgcgcgtttcccggaattccgcgacgagaacatccgcgccgtggctatcgaaaacctgaaaaaacgtgg 16GGCCTGATAAGCGAAGCGCATCAGGCATTTTTGCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCA 17 AACTCATTTCGTTTTTAGGGAGGAATAA 18CTTCCCCTTGAACGGGAGGGCATTTTTCTGAAATATCCTTTCTTTAGCCCATAATAATATTTCCTTTGCTGCGATTTTTTCAATTTCCGATATATTCATAATTTATCAAGGTTGATATAAATATCAGTGAAGATCTCCAGATATTGTTGCGGAACTGGCTACGATAAAAGATAAATCAGA TGATGAATGGTGGCGTGCATTG 19catcctgatgactggtgaactggcagcgttaattcaaaacctgattgaaggattaggtggcgaagcacaacgttaattgctgattttcctttaatgccggatgcgacgcctgccgcgtcttatccggcgtacgaagccacaccaggcatataattattcgctacggcgagcaataatttttagcgcagcaatattatgcgttttacgctgtaacttgctccatggacgttgtgtcattgtttacctcaagccg 20ATGAGTACTGAAATCAAAACTCAGGTCGTGGTACTTGGGGCAGGCCCCGCAGGTTACTCCGCTGCCTTCCGTTGCGCTGACATATGAATA TCCTCCTTAG 21TTACTTCTTCTTCGCTTTCGGGTTCGGCAGGTCGGTAATGCTACCTTCGAACACTTCTGCCGCCAGGCCCACAGACTCGTTGTAGGCTGG AGCTGCTTCG 22TGGCAAGGTAAGCAAACTATAAGGAGGTCAAAT 23aagctttgatggcaagcgataccattcgtattccgggtattgatacaccgctgagccgtgttgcactgggcacctgggcaattggtggttggatgtggggtggtccggatgatgataatggtgttcgtaccattcatgcagcactggatgaaggtattaatctgattgataccgctccggtttatggttttggtcatagcgaagaaattgttggtcgtgcactggcagaaaaaccgaataaagcacatgttgcaaccaaactgggtctgcattgggttggtgaagatgagaaaaacatgaaagtgtttcgtgatagccgtccggcacgtattcgtaaagaagttgaagatagcctgcgtcgtctgcgtgttgaaaccattgatctggaacaaattcattggcctgatgataaaaccccgattgatgaaagcgcacgtgaactgcagaaactgcatcaggatggtaaaattcgtgccctgggtgttagcaattttagtccggaacaaatggatatattcgtgaagttgcaccgctggcaaccattcagcctccgctgaacctgtttgaacgtaccattgaaaaagatattctgccgtatgccgaaaaacataatgcagttgttctggcatatggtgcactgtgtcgtggtctgctgaccggcaaaatgaatcgtgataccacctttccgaaagatgatctgcgtagcaatgatccgaaatttcagaaaccgaacttcgagaaatatctggctgcaatggatgagtttgaaaaactggccgagaaacgtggtaaaagcgttatggcatttgcagttcgttgggttctggatcagggtccggttattgcactgtggggtgcacgtaaaccgggtcaggttagcggtgttaaagatgtttaggttggagcctgaccgacgaagaaaaaaaagcagttgatgatattctggcacgtcatgttccgaatccgattgatccgacctttatggcaccg cctgcacgtgattaagaattc 24aagctttgatggcctccaagacgtacactctgaacaccggtgccaagatacccgcggtcgggttcggcacattcgccaatgagggtgccaagggcgagacatacgcagctgttacaaaggcactggacgttggataccgccaccttgattgcgcgtggttttaccacaacgaagatgaggttggtgacgcggtacgcgattactcgcccgccgacccgacgtgaaacgcgaggatctcttcatttgcaccaaagtaggaaccacctgcatgagccagaggacgtcaagtggagcgccaagaactcgtgcgaaaacctcaaggtcgattacattgacctgttcctcgtccactggccaatcgcggccgagaagaacagcgacaggagcgtcaagctgggccccgatggcaagtatgtcatcaaccaagccctgacggaaaacccagagccaacatggcgagccatggaagagcttgttgaaagcggcctcgtcaaggcaattggagtatccaactggacgattccggggttgaagaagctccttcagatcgccaagatcaagccggcagtgaaccagattgagattcacccattcctaccaaacgaagagcttgtggcgttctgctagagaacgggatcctgcccgaagcctactcgccgctgggctcgcagaaccaggtcccaagcaccggcgagcgagtgcgcgacaacccgacactcaaagcggttgccgagcgaagcggctacagccttgcccagatcctattggcatggggcctgaagcgaggatatgtggtcctcccaaagagctcaactccaagccgtattgaaagcaacttcaacattccggagctgagtgatgaagactagaggcgattcaacaggttgctaaggggagacatactagatttgtcaacatgaaggacacgtaggatacaacgtttgg ccagaggaggaataagaattc 25aagctttgactagtatgaaaggacttgctatgcttggaattggaagaattggatggattgaaaagaaaatcccagaatgtggaccacttgatgcattagttagaccattagcacttgcaccatgtacatcagatacacataccgtttgggcaggagctattggagatagacatgatatgattcttggacatgaagcggttggacaaattgttaaagttggatcattagttaagagattaaaagttggagataaagttattgtaccagctattacaccagattggggagaagaagaatcgcaaagaggatatccaatgcattcaggaggaatgcttggaggatggaaattctcaaatttcaaggatggagttttttcagaagttttccatgttaatgaagcagatgccaatcttgcacttcttccaagagatattaaaccagaagatgcagttatgttatcagatatggtaactactggattccatggagcagaattagctaatattaaacttggagatactgtagtgttattggtattggaccagttggattaatgtcagttgcaggagcaaaccatcttggagcaggaagaatattgcagtaggatcaagaaaacattgttgtgatattgcattggaatatggagcaacagatattattaattataaaaatggagatattgtagaacaaattcttaaagctacagacggcaaaggagttgataaagtcgttattgcaggaggtgatgttcatacatttgcacaagcagtcaaaatgattaaaccaggatcagatattggaaatgttaattatcttggagaaggagataatattgatattccaagaagtgaatggggagttggaatgggtcataaacacattcatggaggtttaaccccaggtggaagagtcagaatggaaaaattagcatcacttatttcaactggtaaattagatacttctaaacttattacacatagatttgaaggattagaaaaagttgaagatgcattaatgttaatgaagaataaaccagcagaccttatcaaaccagttgtcagaattcattatgatgatgaa gatactcttcattaactcgag 26atgaacaactttaatctgcacaccccaacccgcattctgtaggtaaaggcgcaatcgctggtttacgcgaacaaattccgTGTAGGCTGG AGCTGCTTCG 27ttagccgccgaactggtcaggatcgggaccgagacgcttgccctgatcgagttagcaatttcgccgagttcgtctttgCATATGAATATC CTCCTTAGProtocol 5: Evaluation of 1,2-propanediol Production Strains

1,2-propanediol production strains were cultivated in flask cultures asdescribed in patent application EP 2532751, except that 20 g/L glucoseor sucrose and 40 g/L MOPS were used. When necessary 100 μM IPTG wasadded to the medium. 1,2-propanediol (PG) and hydroxacetone (HA) werequantified by HPLC. The production of PG (gPG/L) and the conversion ofHA to PG (gPG/L/(gHA/L+gPG/L)) give an estimate of strain performancefor the production of 1,2-propanediol.

Protocol 6: Flask Cultures for the Production of Recombinant Proteins

Flask cultures for the production of recombinant proteins were carriedout as described in patent application WO 2010/076324 except that the LBbroth was supplemented with 5,0 g/L glucose.

Example 2 Construction of Strains 1, 2, 3 and 4 Construction of Strain 1

To inactivate the fumarate reductase flavoprotein complex encoded by thefrdABCD operon and the glucose phophotransferase Enzyme IIBC(Glc)encoded by the ptsG gene, the homologous recombination strategy was used(according to Protocols 1 and 3). Oligonucleotides for DfrdABCD: SEQ IDNo 1 and 2 (listed in Table 2), and DptsG: SEQ ID No 3 and 4 (listed inTable 2), were used to PCR amplify the resistance cassettes. The strainsretained were designated MG1655 DfrdABCD::Cm and MG1655 DptsG::Km.Finally, the DfrdABCD::Cm and the DptsG::Km deletions were transferredby P1 phage transduction (according to Protocol 2) into the evolvedstrain MG1655 1pd*DtpiA DpflAB DadhE DldhA DgloA DaldA DaldB Dedd DarcADndh described in patent application WO2008/116852, giving rise tostrain 1.

Construction of Strain 2

The gldA*(A160T) gene was cloned into the pME101VB06 plasmid asdescribed in patent application EP 2532751. This plasmid named pPG0078was transformed into strain 1, giving rise to strain 2.

Construction of Strain 3

To express the triose phosphate isomerase encoded by the tpiA gene andto regulate the expression of the glyceraldehyde phosphate dehydrogenaseencoded by the gapA gene, the homologous recombination strategy was used(according to Protocols 1 and 3). The tpiA gene was introduced asdescribed in patent WO2008/116852 into the evolved strain MG16551pd*DtpiA DpflAB DadhE DldhA DgloA DaldA DaldB Dedd DarcA Dndh DfrdABCD.Then the genomic modification to regulate the gapA expression“CI857-PR01/RBS11-gapA” was introduced as described in patent EP 2532751into the previous strain to give rise to strain 3.

Construction of Strain 4

To allow the growth of Escherichia coli on sucrose, the genes scrK,scrYAB and scrR from the plasmid pUR400 (Schmid et al., 1982) werecloned under their natural promoters on the plasmid pBBR1MCS3. Thisplasmid was named pPG0231. Plasmids pPG0078 and pPG0231 were transformedinto strain 3, giving rise to strain 4.

Example 3 Production of 1,2-propanediol is Improved in StrainsOverexpressing the Secondary Alcohol Dehydrogenase adh from Clostridiumbeijerinckii Construction of Strain 5

The adh gene from Clostridium beijerinckii (Hanai et al., 2007) wascloned into the pME101VB01 plasmid described in patent application WO2008/116853. This plasmid named pPG0468 was transformed into strain 1,giving rise to strain 5.

Construction of Strain 6

To inactivate the gldA gene, the homologous recombination strategy wasused (according to Protocols 1 and 3). Oligonucleotides for DgldA: SEQID N° 5 and 6 (listed in Table 2), were used to PCR amplify theresistance cassette. The strain retained was designated MG1655DgldA::Cm. The DgldA::Cm deletion was transferred by P1 phagetransduction (according to Protocol 2) into strain 5, giving rise tostrain 6.

Construction of Strain 7

Plasmids pPG0468 and pPG0231 were transformed into strain 3, giving riseto strain 7.

Construction of Strain 8

To inactivate the gldA gene, the DgldA::Cm deletion previously describedwas transferred by P1 phage transduction (according to Protocol 2) intostrain 7, giving rise to strain 8.

Strains 5, 6, 7 and 8 expressing adh from Clostridium beijerinckiievaluated as described in Protocol 5 produced more 1,2-propanediol (PG)and had better conversion rates and yields compared to their respectivecontrols strains 2 and 4 (Table 3).

TABLE 3 1,2-propanediol production by strains 5, 6, 7 and 8 expressingadh from Clostridium beijerinckii. Culture Strain Control strainconditions PG Conversion Yield 5 2 Glucose 37° C. + + + 6 2 Glucose 37°C. ++ ++ ++ 7 4 Sucrose 37° C. ++/+++ ++/+++ ++/+++ Sucrose 30° C. + + +8 4 Sucrose 37° C. ++++ ++++ ++++ Sucrose 30° C. + + + (The symbol ~indicates that there is no significant difference between the strains,the symbol + indicates an increase between 10 to 100% in performancecompared to the control strain, the symbol ++ indicates an increasebetween 100 to 200% compared to the control strain, the symbol +++indicates an increase between 200 to 300% compared to the control strainand the symbol ++++ indicates an increase greater than 300% compared tothe control strain):

Example 4 Production of 1,2-propanediol is Improved by Overexpressingthe Pyridine Nucleotide Transhydrogenase pntAB in Strains Overexpressingthe Secondary Alcohol Dehydrogenase adh from Clostridium beijerinckiiConstruction of Strain 9

The native promoter region of pntAB was replaced by the inducible trcpromoter (from the plasmid pTRC99A, Amersham Pharmacia) and the defineribosome binding site RBS120 (from RBS Calculator software) (SEQ ID No 7listed in Table 2) using the homologous recombination strategy(according to Protocols 1 and 3). For chromosomal integration, afragment carrying the artificial promoter region and a resistance markerboth flanked by homologous DNA sequences to the targeted integrationlocus pntAB was PCR amplified by overlapping PCR technique (overlappingoligonucleotides). The sequences for recombination into pntAB arereferred to as SEQ ID No 8 and 9 (listed in Table 2). The PCR productobtained was then introduced by electroporation into the strain MG1655(pKD46). The strain retained was designated MG1655Ptrc01/OP01/RBS120-pntAB::Cm. Then, the Ptrc01/OP01/RBS120-pntAB::Cmmodification was transferred by P1 phage transduction (according toProtocol 2) into strain 6, giving rise to strain 9.

Construction of Strain 10

The Ptrc01/OP01/RBS120-pntAB::Cm modification previously described wastransferred by P1 phage transduction (according to Protocol 2) intostrain 8, giving rise to strain 10.

Strains 9 and 10 overexpressing pntAB evaluated as described in Protocol5 produced more 1,2-propanediol (PG) and had better conversion rates andyields compared to strains 6 and 8, respectively (Table 4).

TABLE 4 1,2-propanediol production by strains 9 and 10 overexpressingpntAB. Culture Strain Control strain conditions PG Conversion Yield 6 2Glucose 37° C. ++ ++ ++ 9 2 Glucose 37° C. +++ +++ +++ 8 4 Sucrose 30°C. + + + 10 4 Sucrose 30° C. +++ +++ +++ (The symbol ~ indicates thatthere is no significant difference between the strains, the symbol +indicates an increase between 10 to 100% in performance compared to thecontrol strain, the symbol ++ indicates an increase between 100 to 200%compared to the control strain, the symbol +++ indicates an increasebetween 200 to 300% compared to the control strain and the symbol ++++indicates an increase greater than 300% compared to the control strain):

Example 5 Alternatives to the Overexpression of pntAB to Improve1,2-propanediol Production in Strains Overexpressing the SecondaryAlcohol Dehydrogenase adh from Clostridium beijerinckii Construction ofStrain 11

The yjeF gene from Escherichia coli was cloned in operon with adh underthe define ribosome binding site RBS121 (from RBS Calculator software)(SEQ ID No 10 listed in Table 2) into the pPG0468 plasmid described inexample 3. This plasmid was named pPG0518. Finally the plasmids pPG0518and pPG0231 were transformed into the intermediate strain 8 (withoutplasmid) giving rise to strain 11.

Construction of Strain 12

To inactivate the pgi gene, the homologous recombination strategy wasused (according to Protocols 1 and 3). Oligonucleotides for Dpgi: SEQ IDNo 11 and 12 (listed in Table 2), were used to PCR amplify theresistance cassette. The strain retained was designated MG1655 Dpgi::Cm.The Dpgi::Cm deletion was transferred by P1 phage transduction(according to Protocol 2) into strain 8, giving rise to strain 12.

Construction of Strain 13

The native pfkA gene was replaced by the mutated pfkA*(L98Q) gene usingthe homologous recombination strategy (according to Protocols 1 and 3).First the pfkA gene was deleted using the oligonucleotides SEQ ID No 13and 14 (listed in Table 2) to PCR amplify the resistance cassette. Thestrain retained was designated MG1655 DpfkA::Km. Then, for chromosomalintegration, a fragment carrying the mutated pfkA*(L98Q) region and aresistance marker both flanked by homologous DNA sequences to thetargeted integration locus pfkA was PCR amplified by overlapping PCRtechnique (overlapping oligonucleotides). The sequences forrecombination into pfkA are referred to as SEQ ID No 15 and 16 (listedin Table 2). The PCR product obtained was then introduced byelectroporation into the strain MG1655 DpfkA::Km (pKD46). The strainretained was designated MG1655 pfkA*(L98Q)::Cm. The pfkA*(L98Q)::Cmmodification was transferred by P1 phage transduction (according toProtocol 2) into the intermediate strain 8 (strain without plasmid). Thezwf gene from Escherichia coli was cloned in operon with adh under thedefine ribosome binding site RBS113 (from RBS Calculator software) (SEQID No 17 listed in Table 2) into the pPG0468 plasmid described inexample 3. This plasmid was named pPG0532. Finally the plasmids pPG0532and pPG0231 were transformed into the previous strain giving rise tostrain 12.

Construction of Strain 14

To inactivate the gldA gene, the DgldA::Cm deletion previously describedwas transferred by P1 phage transduction (according to Protocol 2) intothe intermediate evolved strain 3 (without gapA regulation). To expressthe NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase fromStreptococcus mutans encoded by the gapN gene, the homologousrecombination strategy was used (according to Protocols 1 and 3). ThegapA gene from Escherichia coli was replaced by the gapN gene fromStreptococcus mutans as described by Centeno-Leija et al. (2013). ThePCR product obtained was introduced by electroporation into the strainMG1655 (pKD46). The strain retained was designated MG1655 gapN::Cm. ThegapN::Cm modification was transferred by P1 phage transduction(according to Protocol 2) into the previous strain. To chromosomallyexpress the genes scrK, scrYAB and scrR, the homologous recombinationstrategy described by Datsenko & Wanner, 2000 (according to Protocols 1and 3) was used. For chromosomal integration, a fragment carrying thegenes scrK, scrYAB and scrR expressed under their natural promoterslinked to a resistance marker both flanked by homologous DNA sequencesto the targeted integration locus ykiA was PCR amplified by overlappingPCR technique (overlapping oligonucleotides). The sequences forrecombination into ykiA are referred to as SEQ ID No 18 and 19 (listedin Table 2). The PCR products “DykiA::scrKYABR” obtained was thenintroduced by electroporation into the strain MG1655 (pKD46). The strainretained was designated MG1655 DykiA::scrKYABR::Cm. TheDykiA::scrKYABR::Cm modification was transferred by P1 phagetransduction (according to Protocol 2) into the previous strain. Toallow the overproduction of the NADP+-dependent glyceraldehyde3-phosphate dehydrogenase from Streptococcus mutans, the gapN gene wascloned on the pACYC184 plasmid as described by Centeno-Leija et al.(2013). This plasmid was named pPG0548. Finally the plasmids pPG0468 andpPG0548 were transformed into the previous strain giving rise to strain13.

Construction of Strain 15

To inactivate the NAD+-dependent lipoamide dehydrogenase encoded by thelpd gene, the homologous recombination strategy was used (according toProtocols 1 and 3). Thus the oligonucleotides for Dlpd: SEQ ID N° 20 and21 (listed in Table 2), were used to PCR amplify the resistancecassette. The strain retained was designated MG1655 Dlpd::Cm. Finally,the Dlpd::Cm deletion was transferred by P1 phage transduction(according to Protocol 2) into the intermediate strain 8, (strainwithout plasmid). The mutated1pd*(A55V/G185A/G189A/E203V/M204R/F205K/D206H/P210R) gene fromEscherichia coli was PCR amplified by overlapping PCR technique(overlapping oligonucleotides) and cloned in operon with adh under thedefine ribosome binding site RBS131 (from RBS Calculator software) (SEQID No 22 listed in Table 2) into the pPG0468 plasmid described inexample 3. This plasmid was named pPG0523. Finally the plasmids pPG0231and pPG0523 were transformed into the previous strain giving rise tostrain 14.

Strains 11, 12, 13, 14 and 15 evaluated as described in Protocol 5produced more 1,2-propanediol (PG) and had better conversion ratescompared to strain 8 (Table 5).

TABLE 5 1,2-propanediol production by strains 11, 12, 13, 14 and 15.Culture Strain Control strain conditions PG Conversion 8 4 Sucrose 30°C. + + 11 4 Sucrose 30° C. ++ ++ 12 4 Sucrose 30° C. +++ +++ 13 4Sucrose 30° C. +++ +++ 14 4 Sucrose 30° C. +++ +++ 15 4 Sucrose 30° C.+++ +++ (The symbol ~ indicates that there is no significant differencebetween the strains, the symbol + indicates an increase between 10 to100% in performance compared to the control strain, the symbol ++indicates an increase between 100 to 200% compared to the controlstrain, the symbol +++ indicates an increase between 200 to 300%compared to the control strain and the symbol ++++ indicates an increasegreater than 300% compared to the control strain):

Example 6: Alternatives to adh from Clostridium beijerinckii

Identification of Homologous Sequences and Construction of PhylogeneticTrees

The constructions of phylogenetic trees from a set of sequences wereperformed using the bioinformatics programs available on the websitehttp://www.phylogeny.fr/ and the softwares are described in Dereeper etal. (2008) and Dereeper et al. (2010).

Step 1: Identification of Homologous Sequences

The identification of similar sequences was performed from secondaryalcohol dehydrogenase family and aldehyde/ketone reductase family usingthe BLAST software by setting the following parameters: database(Swissprot/UNIPROT), e-value=0.01 and filter for low-complexitysequences.

Step 2: Construction of Phylogenetic Trees

Sequences were aligned with MUSCLE software and ambiguous regions wereremoved with Gblocks programm. Then, the phylogeneic tree wasreconstructed using the maximum likelihood method implemented in thePhyML program. Graphical representation and edition of the phylogenetictree were performed with TreeDyn.

TABLE 6 Candidates for NADPH dependent HAR enzymes: Organism GI UNIPROTClostridium _(—) beijerinckii 166228784 P25984 Thermoanaerobacter _(—)brockii 113443 P14941 Mycoplasma _(—) pneumoniae 2492770 P75214Entamoeba _(—) histolytica 543776 P35630 Gadus _(—) callarias 113437P26325 Photobacterium _(—) damselae 728805 P39450 Synechocystis_sp6225006 P73138 Oryza _(—) sativa 75339814 Q4R1E8 Hordeum _(—) vulgare113385 P10848 Sparus _(—) aurata 5902742 P79896 Gadus _(—) morhua5902740 P81600 Hordeum _(—) vulgare 113376 P10847 Zea _(—) mays 113377P04707 Saccharomyces _(—) cerevisiae 586489 P38230 Pennisetum _(—)glaucum 113362 P14219 Oryctolagus _(—) cuniculus 3912977 O46650Arabidopsis _(—) thaliana 122223583 Q0V7W6 Uromastyx _(—) hardwickii1351882 P25405 Amycolatopsis _(—) methanolica 3915692 P80094 Zea _(—)luxurians 1351874 Q07264 Zea _(—) mays 113359 P00333 Oryctolagus _(—)cuniculus 3912976 O46649 Arabidopsis _(—) thaliana 148841208 P06525Arabidopsis _(—) thaliana 158563847 Q8LEB2 Cupriavidus _(—) necator113411 P14940 Rana _(—) perezi 113364 P22797 Arabidopsis _(—) thaliana75337342 Q9SK87 Rattus _(—) norvegicus 48474724 Q64563 Malus _(—)domestica 1351887 P48977 Drosophila _(—) melanogaster 1168359 P46415 Mus_(—) musculus 338817905 Q9QYY9 Homo _(—) sapiens 308153684 P08319Bacillus _(—) subtilis 3915097 O31776 Peromyscus _(—) maniculatus1168352 P41681 Bacillus _(—) licheniformis 67461713 Q65JE7 Arabidopsis_(—) thaliana 75337341 Q9SK86 Aspergillus _(—) fumigatus 294863186B0YC65 Saccharomyces _(—) cerevisiae 417769 P32771 Staphylococcus _(—)aureus 81827907 Q6GBM4 Staphylococcus _(—) aureus 81832555 Q7A742Aspergillus _(—) oryzae 74696959 Q86ZV0 Aspergillus _(—) clavatus294863185 A1CFY8 Sulfolobus _(—) tokodaii 74580971 Q96XE0 Neosartorya_(—) fischeri 294863179 A1D9C9 Metallosphaera _(—) sedula 353678127A4YGN0 Sulfolobus_sp 1703176 P50381 Bacillus _(—) subtilis 461928 Q06004Escherichia _(—) coli 2492775 P77280 Saccharomyces _(—) cerevisiae74655020 Q07993 Thermococcus _(—) kodakarensis 67461628 Q5JI69Methylobacter _(—) marinus 221222447 P47734 Bacillus _(—) subtilis81815843 O35045 Bacillus _(—) subtilis 251757237 O06012Schizosaccharomyces _(—) pombe 74698317 Q9P6I8 Escherichia _(—) coli2492774 P75691 Mycobacterium _(—) tuberculosis 81815028 O07737Saccharomyces _(—) cerevisiae 2492777 Q04894 Streptomyces _(—)tenebrarius 94710637 Q2MF22 Escherichia _(—) coli 732035 P27250 Bacillus_(—) subtilis 3123233 P80874 Gluconobacter _(—) oxydans 81557013 Q5FQJ0Hypocrea _(—) jecorina 121924008 Q0GYU4 Dunaliella salina 219107912B8Y210

Adh from Clostridium beijerinckii was most closely related to theThermoanaerobacterium brockii, Entamoeba histolytica and Mycoplasmapneumoniae enzymes. The next best match was with alcohol dehydrogenasesfound in eukaryotes. Enzymes from Gluconobacter oxydans, Hypocreajecorina, Entamoeba histolytica and Bacillus subtilis were cloned,purified and assayed for NADPH dependent hydroxyacetone reductaseactivity.

Construction of Strain 16

To inactivate the gldA gene, the homologous recombination strategy wasused (according to Protocols 1 and 3). Oligonucleotides for DgldA: SEQID No 5 and 6 (listed in Table 2), were used to PCR amplify theresistance cassette. The strain retained was designated MG1655DgldA::Km. Finally, the DgldA::Km deletion was transferred by P1 phagetransduction (according to Protocol 2) into the strain BL21(DE3)star. Tocharacterize the glycerol dehydrogenase from Gluconobacter oxydans, thesynthetic gene gld optimized for Escherichia coli (SEQ ID No 23 listedin table 2) was cloned into the expression plasmid pPAL7 (Biorad®). Thisplasmid was named pPG0381 and transformed into strain BL21(DE3)starDgldA::Km, giving rise to strain 16.

Construction of Strain 17

To characterize the glycerol dehydrogenase from Hypocrea jecorina, thesynthetic gene gld2 optimized for Escherichia coli (SEQ ID No 24 listedin table 2) was cloned into the expression plasmid pPAL7 (Biorad®). Thisplasmid was named pPG0418 and transformed into strain BL21(DE3)starDgldA::Km previously described giving rise to strain 17.

Construction of Strain 18

To characterize the alcohol dehydrogenase from Entamoeba histolytica,the synthetic gene adh1 optimized for Escherichia coli (SEQ ID No 25listed in table 2) was cloned into the expression plasmid pPAL7(Biorad®). This plasmid was named pPG0539 and transformed into strainBL21(DE3)codonplus, giving rise to strain 18.

Construction of Strain 19

To characterize the aldo keto reductase from Bacillus subtilis, the geneyhdN from Bacillus subtilis was cloned into the expression plasmid pPAL7(Biorad®). This plasmid was named pPG0357 and transformed into strainBL21(DE3), giving rise to strain 19.

Recombinant Protein Purification

Strains 16, 17, 18 and 19 were cultivated as described in Protocol 6.Cells (350-500 mg dry weight) were resuspended in extraction buffer(60-90 ml) with protease inhibitor cocktail. The suspended cells weredisrupted by six sonication cycles of 30 sec on ice (Branson sonifier,70W) followed by incubating for 1 hour at room temperature with 1 mMMgCl2 and 2UI/ml of DNasel. Cell debris were removed by centrifugationat 12000 g for 30 min at 4° C. The supernatant was kept as the crudecell-extract._Recombinant protein was purified from the crude extract byusing subtilisine affinity chromatography (PROfinity EXact Cartridge 5ml, BIORAD) according to the manufacturer's instructions. Fractionscontaining the protein were pooled, concentrated and loaded onto a gelfiltration column (Superdex 200 10/300 GL column, GE Healthcare).Protein concentration was determined using Bradford assay.

NADPH Dependent Hydroxyacetone Reductase (HAR NADPH) of Purified Enzymes

HAR NADPH activity was determined by measuring the consumption of NADPHat 340 nm on a spectrophotometer (ε_(340nm)=6290 M⁻¹ cm⁻¹) and at 30° C.The reaction mixture (1 mL) containing assay buffer, 0.2 mM NADPH andpurified protein was incubated for 5 min at 30° C. Then, 30 mMhydroxyacetone was added to start the reaction. One unit of enzymeactivity was defined as the amount of enzyme catalyzing the decrease of1 μmol of NADPH per min. Specific enzyme activity was expressed as unitsof enzyme activity per mg of protein. The activity value determinedwithout hydroxyacetone was subtracted.

TABLE 7 HAR NADPH dependent activity for specific candidates: HAR NADPHEnzyme Assay buffer (mUI/mg) Gld 20 mM Hepes (pH 7.5) 5461 Gld2 10 mMsodium phosphate (pH 7) 9623 Adh1 50 mM Tris-HCl (pH 7.5) 13325 YhdN 20mM Tris-HCl (pH 7.5) 1975

Construction of Strain 20

The optimized adhl gene from Entamoeba histolytica was cloned into thepME101VB01 plasmid described in patent application WO 2008/116853. Thisplasmid was named pPG0544. Plasmids pPG0544 and pPG0231 were transformedinto strain 3 described in Example 2, then the gldA gene was inactivatedas described in Example 3 and the pntAB operon was overexpressed asdescribed in Example 4, giving rise to strain 19.

Strain 20 evaluated as described in Protocol 5 produced more1,2-propanediol (PG) and had a better conversion rate compared to strain4 (Table 8).

TABLE 8 1,2-propanediol production by strain 20 expressing adh1 genefrom Entamoeba histolytica. Culture Strain Control strain conditions PGConversion 20 4 Sucrose 37° C. +++ +++ (The symbol ~ indicates thatthere is no significant difference between the strains, the symbol +indicates an increase between 10 to 100% in performance compared to thecontrol strain, the symbol ++ indicates an increase between 100 to 200%compared to the control strain, the symbol +++ indicates an increasebetween 200 to 300% compared to the control strain and the symbol ++++indicates an increase greater than 300% compared to the control strain):

Example 7: Co-Factor Refactoring of G1dA*(A160T)

Enzyme Design and Structural Analysis

Homology models of GldA*(A160T) were built from the X-ray structure of aglycerol dehydrogenase of Bacillus stearothermophilus. The models werecalculated by using the Discovery Studio software (Accelrys). Thesemodels were compared with the structure of NAD and NADP dependentenzymes which had at least 30% identity and whose structure was known.

Amino acids involved in cofactor specificity were identified throughsequence alignment between the sequence of GldA*(A160T) and several NADand NADP dependent dehydrogenases, superimposition between the homologymodels of GldA*(A160T) and the glycerol dehydrogenases present in theRCSB Protein Data Bank, and comparison with data found in Clermont etal. (1993), Ruzheinikov et al. (2001), Corbier et al. (1990) and Wu etal. (2012).

Two mutants were defined from the sequential and structural analysis:D37G and D37G/P161S/L164A.

Construction of Strain 21

To characterize the mutated 1,2-propanediol:NAD+ oxidoreductase fromEscherichia coli, first the gene gldA was cloned into the expressionplasmid pET101/D-TOPO (Lifetechnologies®). This plasmid was namedpPG0029. To overexpress the mutated gldA*(A160T) gene, site-directedmutagenesis on pPG0029 was used. This plasmid was named pPG0394 andtransformed into strain BL21(DE3)star DgldA::Km, giving rise to thestrain 21.

Construction of Strain 22

To overexpress the mutated gldA*(A160T/D37G) gene, site-directedmutagenesis on pETTOPO-gldA*(A160T) was used. This plasmid was namedpPG0425 and transformed into strain BL21(DE3)star DgldA::Km, giving riseto the strain 22.

Construction of Strain 23

To overexpress the mutated gldA*(A160T/D37G/P161S/L164A) gene,site-directed mutagenesis on pPG0425 was used. This plasmid was namedpPG0438 and transformed into strain BL21(DE3)star DgldA::Km, giving riseto the strain 23.

Purification of GldA*(A160T), GldA*(A160T/D37G) andGldA*(A160T/D37G/P161S/L164A)

Strains 21, 22 and 23 were cultivated as described in Protocol 6. Cells(500 mg dry weight) were resuspended in 90 mL of extraction buffer(100mM potassium phosphate pH 7.6, 20 mM imidazole and proteaseinhibitor cocktail). The suspended cells were disrupted by 8 sonicationcycles of 30 sec on ice (Branson sonifier, 70W) followed by anincubation for 45 min at room temperature with 5 mM MgC12 and 2 UI/ml ofDNasel. Cell debris were removed by centrifugation at 12000 g for 30 minat 4° C. The supernatant was kept as the crude extract._The enzymes werepurified from the crude extract by using Nickel affinity chromatography(HisTrapFF 1 mL, GE Healthcare) according to the manufacturer'sinstructions. The enzymes were eluted by using a linear gradient ofimidazole (20 to 500 mM) in 100 mM potassium phosphate (pH 7.6). After adesalting step by gel filtration (Superdex200 10/300 GL column, GEHealthcare) equilibrated with 100mM MES-KOH (pH 6.5), proteinconcentrations were determined using Bradford assay.

Characterization of GldA*(A160T), GldA*(A160T/D37G) andGldA*(A160T/D37G/P161S/L164A)

NADPH dependent hydroxyacetone reductase activity (HAR NADPH) wasdetermined by measuring the consumption of NADPH at 340 nm on aspectrophotometer (

₃₄₀=6290 M⁻¹ cm⁻¹) and at 30° C. The reaction mixture (1 mL) containing100 mM MES-KOH (pH 6.5), 0.1 mM FeSO4, 30 mM ammonium sulfate, 0.05 to0.4 mM NADPH and purified enzyme was incubated for 5 min at 30° C. Then,0.1 to 10 mM hydroxyacetone was added to start the reaction. The NADHdependent hydroxyacetone reductase (HAR NADH) assay is performed underthe same conditions that HAR NADPH except that NADPH was replaced byNADH in the reaction mixture and the activity was determined bymeasuring the consumption of NADH at 340 nm. Kinetic parameters weredetermined with Sigmaplot by fitting to the Michaelis-Menten equation.

GldA*(A160T/D37G) and GldA*(A160T/D37G/P161S/L164A) enzymes showedincreased catalytic efficiencies with NADPH and decreased catalyticefficiencies with NADH compared to GldA*(A160T) (Table 9).

TABLE 9 Catalytic efficiencies of GldA* NADPH mutants: GldA* GldA* GldA*Enzyme (A160T) (A160T/D37G) (A160T/D37G/P161S/L164A) Kcat/Km 10 177 168NADPH (mM−1s−1) Kcat/Km 98 61 41 NADH (mM−1s−1)

Construction of Strain 24

The mutated gldA*(A160T/D37G/P161S/L164A) gene was cloned into thepME101 derived plasmid as described for the construction ofpME101VB06-gldA*(A160T) in patent application EP 2532751. This plasmidwas named pPG0467. Finally the plasmids pPG0467 and pPG0231 weretransformed into the intermediate strain 8 (strain without plasmid)described in example 2, giving rise to strain 24.

Strain 24 evaluated as described in Protocol 5 produced more1,2-propanediol (PG) and had a better conversion rate compared to strain4 (Table 10).

TABLE 10 1,2-propanediol production by strain 24. Culture Strain Controlstrain conditions PG Conversion 24 4 Sucrose 37° C. +++ ++ (The symbol ~indicates that there is no significant difference between the strains,the symbol + indicates an increase between 10 to 100% in performancecompared to the control strain, the symbol ++ indicates an increasebetween 100 to 200% compared to the control strain, the symbol +++indicates an increase between 200 to 300% compared to the control strainand the symbol ++++ indicates an increase greater than 300% compared tothe control strain):

Example 8 MG→PG activity by Adh from Clostridium beijerinckiiConstruction of Strain 25

The optimized adh gene from Clostridium beijerinckii (Hanai, Atsumi andLiao, 2007) was cloned into the expression plasmid pET28a. This plasmidwas named pPG0445 and transformed into strain BL21(DE3), giving rise tostrain 25.

Purification of Adh from Clostridium beijerinckii

Strain 25 was cultivated as described in Protocol 6. Cells (315 mg dryweight) were resuspended in 50 mL of extraction buffer (20 mM Tris-HClpH 7.3, 0.1 mM DTT, 0.1 mM benzamidine, 10% glycerol, 0.02% sodium azideand protease inhibitor cocktail). The suspended cells were disrupted by8 sonication cycles of 30 sec on ice (Branson sonifier, 70W). Celldebris were removed by centrifugation at 12000 g for 30 min at 4° C. Thesupernatant was exposed to heat (65° for 5 min) and then recentrifugedat 12000g for 30 min at 4° C._The protein was purified from thesupernatant by using Nickel affinity chromatography (HisTrapFF 1 mL, GEHealthcare) according to the manufacturer's instructions. The proteinwas eluted by using a linear gradient of imidazole (20 to 500 mM) in 50mM Tris-HC1 (pH 7.4). The fractions which contain the protein werepooled, concentrated and dialysed against 50 mM Tris-HCl (pH 7). Proteinconcentration was determined using Bradford assay.

Quantification of 1,2-propanediol Produced from Methylglyoxal

5-10 μg of purified enzyme was incubated for 30 min at 30° C. in 50 mMTris-HCl (pH7.5), 10 mM methylglyoxal and 5 mM NADPH. The quantity of1,2-propanediol produced by Adh from methylglyoxal was measured directlyby GC-MS (Agilent Technologies).

Under such conditions 4.6 mM of 1,2-propanediol were produced while no1,2-propanediol was produced when either methylglyoxal or enzyme wereomitted.

Construction of Strains 26, 27 and 28

To inactivate the gldA gene, the DgldA::Km deletion previously describedand the tpiA gene were co-transferred by P1 phage transduction(according to Protocol 2) into the strain MG1655 1pd*DtpiA DpflAB DadhEDldhA DgloA DaldA DaldB Dedd described in patent applicationWO2008/116852. The genomic modification to regulate the gapA expression“CI857-PR01/RBS11-gapA” was introduced as described in patent EP 2532751into the previous strain. To inactivate the aldehyde reductase encodedby the yqhD gene and the glyoxal reductase encoded by the yqhE gene, thehomologous recombination strategy was used (according to Protocols 1 and3). Oligonucleotides for DyqhDE: SEQ ID

No 26 and 27 (listed in Table 2) were used to PCR amplify the resistancecassettes. The strain retained was designated MG1655 DyqhDE::Bs. TheDyqhDE::Bs deletion was transferred by P1 phage transduction (accordingto Protocol 2) into the previous strain, giving rise to strain 26. Thenthe plasmid pPG0468 was transformed into this strain, giving rise tostrain 27. Finally the Ptrc01/OP01/RBS120-pntAB::Cm modificationpreviously described was transferred by P1 phage transduction (accordingto Protocol 2) into strain 27, giving rise to strain 28.

When evaluated as described in Protocol 5 strains 27 and 28 producedmore 1,2-propanediol (PG) than strain 26.

REFERENCES:

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1. A method for the production of 1,2-propanediol in a fermentativeprocess comprising the steps: culturing a microorganism geneticallymodified for the production of 1,2-propanediol in an appropriate culturemedium comprising a carbohydrate as source of carbon, and recovering1,2-propanediol from said culture medium, wherein in said geneticallymodified microorganism at least one gene coding for a NADPH dependentacetol reductase or a mutant gldA*gene coding for a NADPH dependentglycerol dehydrogenase is overexpressed, said NADPH dependent acetolreductase having at least 60% amino acids identity with protein encodedby the adh gene from Clostridium beijerinckii or the adh gene fromThermoanaerobacter brockii or the adh1 gene from Entamoeba histolyticaor the GOX1615 gene from Gluconobacter oxydans or the gld2 gene fromHypocrea jecorina or the yhdN gene from Bacillus subtilis.
 2. The methodof claim 1, wherein the NADPH availability is increased in themicroorganism by at least one of the following genetic modifications:the pntAB gene operon coding for a nicotinamide nucleotidetranshydrogenase is overexpressed, and/or the pgi gene coding for aphosphoglucose isomerase is attenuated, and/or the pfkA gene coding fora phosphofructokinase is attenuated, and/or the zwf gene coding for aglucose-6-phosphate dehydrogenase is overexpressed, and/or the yjeF genecoding for an ADP-dependent dehydratase is overexpressed, and/or thegapN gene coding for a NADP-dependent glyceraldehyde-3-phosphatedehydrogenase is overexpressed, and/or a mutant lpd*gene coding for aNADP-dependent lipoamide dehydrogenase is overexpressed.
 3. The methodof claim 1 or 2, wherein the gldA gene coding for a NADH dependentglycerol dehydrogenase is deleted in the microorganism.
 4. The method ofany one of claims 1 to 3, wherein the microorganism further comprisesthe deletion of the yqhD gene coding for a methylglyoxal reductase. 5.The method of any one of claims 1 to 4, wherein the microorganism isselected among the group consisting in Enterobacteriaceae, Bacillaceae,Clostridiaceae, Streptomycetaceae and yeasts.
 6. The method of claim 5,wherein the microorganism is selected among the group consisting ofEscherichia coli, Klebsiella pneumoniae, Thermoanaerobacteriumthermosaccharolyticum, Clostridium sphenoides and Saccharomycescerevisiae.
 7. The method of claim 6, wherein the microorganism is E.coli.
 8. A microorganism genetically modified for the production of1,2-propanediol from a carbohydrate as source of carbon, wherein in saidmicroorganism at least one gene coding for a NADPH dependent acetolreductase or a mutant gldA*gene coding for a NADPH dependent glyceroldehydrogenase is overexpressed, said NADPH dependent acetol reductasehaving at least 60% amino acids identity with protein encoded by the adhgene from Clostridium beijerinckii or the adh gene fromThermoanaerobacter brockii or the adh1 gene from Entamoeba histolyticaor the GOX1615 gene from Gluconobacter oxydans or the gld2 gene fromHypocrea jecorina or the yhdN gene from Bacillus subtilis.
 9. Themicroorganism of claim 8, further comprising at least one of thefollowing genetic modifications: deletion of the gldA gene coding for aNADH dependent glycerol dehydrogenase, and/or deletion of the yqhD genecoding for an aldehyde reductase, and/or deletion of the pgi c genecoding for a phosphoglucose isomerase, and/or deletion of the pjkA genecoding for a phosphofructokinase, and/or overexpression of the pntABgene coding for a nicotinamide nucleotide transhydrogenase, and/oroverexpression of the zwf gene coding for a glucose-6-phosphatedehydrogenase, and/or overexpression of the yjeF gene coding for anADP-dependent dehydratase, and/or overexpression of the gapN gene codingfor a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and/oroverexpression of a mutant lpd*gene coding for a NADP-dependentlipoamide dehydrogenase.
 10. The microorganism of claim 8 or 9, whereinit is selected among the group consisting in Enterobacteriaceae,Bacillaceae, Clostridiaceae, Streptomycetaceae, Corynebacteriaceae andyeasts.
 11. The microorganism of claim 10, wherein it is selected amongthe group consisting of Escherichia coli, Klebsiella pneumoniae,Thermoanaerobacterium thermosaccharolyticum, Clostridium sphenoides andSaccharomyces cerevisiae.